The practice of adding harmless bacterial to food and feed products is well known. Bacteria are also widely used for producing food products by fermentation processes. Typically, such bacteria are of the genera Lactococcus, Streptococcus, Lactobacillus, Bifidobacterium, Enterococcus, Propionibacterium, Leuconostoc, and Pediococcus, depending on the purpose for which the bacteria are cultured. In recent years, especially in the dairy industry, various compositions commonly referred to as "starter" media have been developed to increase the concentration and/or activity of the bacteria in the cultures produced for use in the food or feed industries. (The term "activity" as used herein designates the rate of acid production per unit volume of a bacterial culture.)
In growing dairy starter culture, pH control has been used commercially. As the bacteria ferment lactose to lactic acid, the buffering capacity of the growth medium is eventually overcome; and then the pH drops until acidic conditions become unfavorable for the continued rapid growth of the bacteria. Harvey, J. Bact., 90:1330 (1965) stated that growth in an environment below pH 5.0 results in reduced enzyme activity and cell reproduction. Moreover, extended storage of bacteria in an acidic environment can result in cell damage, reduction in cellular energy reserves, and a loss of activity by the culture. To compensate for such a loss in activity, increased amounts of culture or extended production times are usually required.
The dairy industry in an earlier procedure reduced acid damage by cooling the medium containing the bacteria before the medium reached an inhibitory pH. By dropping the temperature of the medium, bacterial growth and acid production can be essentially stopped. Compositions or media containing high numbers of bacteria are commonly referred to as "ripened starters". A cold ripened starter can be used over a longer period of time than non-cooled starter. This procedure works but is dependent upon the cheesemaker to cool the starter medium at exactly the right time.
Various other methods have been developed to reduce acid damage to the bacteria and increase activity of starter cultures. One method has been referred to as "external pH control." In commercial practice, external pH control has used an electronic pH meter with pH electrodes, a neutralizer, and pump to control the pH. Typically, a pH electrode and meter monitor the pH of the medium in the bulk starter tank. As the bacteria grow in the medium, the pH of the medium drops. When the pH of the medium reaches a preset point, the pump is activated by the meter and neutralizer is added to the tank raising the pH of the medium. The increase in pH of the medium is sensed by the pH meter and the pump is turned off. This cycle is typically repeated many times before the carbohydrate in the medium is exhausted, or before the lactate concentration becomes inhibitory. Ammonium hydroxide is the most commonly used neutralizer, but ammonia gas has also been used. Concentrated solutions of sodium or potassium hydrox-ide have been experimentally tested, but are not known to be used in industrial practice.
A second known method is referred to as "internal pH control". In this method, buffer salts, which are insoluble at higher pH values, dissolve as the bacteria produce acid with the lowering of the pH of the medium. As the salts dissolve, they increase the buffering capacity of the medium and reduce the rate of the pH drop. Sandine U.S. Reissue Patent No. 32,079 describes methods and compositions to "internally" control media pH. See Thunell, "pH-Controlled Starter: A Decade Reviewed," Cultured Dairy Products Journal, August, 1988, pages 10-16).
In external pH control, the acid is neutralized by addition of a base, while in internal pH control the amount of buffer salt is increased by the increasing solubility of the salt as the pH decreases. In an external system, it is not possible to determine the amount of acid produced by the bacteria by titrating with sodium hydroxide to a phenothalein end-point. In an internal pH buffered system, it is possible since the acid is not neutralized.
Reddy, in U.S. Pat. No. 4,622,304, describes a alternative system which can be referred to as "one-step neutralization." (See Thunell, cited above.) In this system, the bacteria are grown until a first predetermined pH or titratable acidity is attained. At that point, a base or basecontaining solution is rapidly added manually to reach a second predetermined pH or titratable acidity. The bacteria are then allowed to continue growing until a third pH or titratable acidity is reached, at which time the medium containing the bacteria is cooled. The bases used are typically solutions of sodium or potassium hydroxide.
The ability of various bacteria to hydrolyze urea by producing an enzyme is known. For example, the Ninth Edition of Bergey's Manual of Systematic Bacteriology published in 1986 at page 1421 describes the ability of Bifidobacteria to hydrolyze urea. [See also Suzuki, et al., Appl. Environ. Microb., 37:379-382 (1979).] Tinson, et al., Austral. J. Dairy Tech., March, 1982, page 14, describes the metabolism of Streptococcus thermophilus, stating that its growth in skim milk decreased acid production midway through the log phase of growth. It was postulated that this apparent decrease in acid production, as monitored by a decrease in pH, was due to urease hydrolysis of a small amount of urea which was naturally present in the milk. This indigenous urea was present at a very low concentration, viz., 1.7 mM or 0.01% on a wt./wt. basis.
Juillard, V., et al., Can. J. Microbiol., 34:818-822 1988), describes the urease enzyme of S. thermophilus. Their study described the effect of temperature, pH, and substrate concentration on the urease activity. Julliard, et al. also found that urease production is strain dependent, viz. some strains produce high levels of urease while other strains produce little or none at all. In their experiments, urea was added to growth media to determine its effect on the stimulation of urease production.
The enzyme urease is produced by a number of bacteria, yeasts, molds, and plants. It is also known by the International Union of Biochemists Number 3.5.1.5. It can be extracted from jack beans, and is commercially used in testing for urea in body fluids. The article by Juillard, et al. (cited above) lists several bacterial sources of urease.